1. Field of the Invention
The present invention relates to methods for visualizing biological materials in gels. More specifically, it relates to the visualization of nucleic acids in polyacrylamide gels.
2. Description of the Related Art
Modern biotechnological techniques require the visualization of very small amounts of biological materials, especially nucleic acids. For example, restriction length fragment polymorphism (RFLP) mapping is a procedure which first divides a length of DNA into hundreds (or thousands) of much smaller fragments. The collection of fragments is then separated by length using electrophoresis. This results in a particular pattern of fragment bands. In general, the bands are not visualized directly but are usually treated with a probe such that at least some of the bands may be seen.
Present techniques for visualization use either hybridization probes or direct visualization. In hybridization techniques, a probe (a radioactive isotope or a fluorescent moiety) is attached or incorporated into a short segment of nucleic acid. The short segment binds to (hybridizes with) any strand of nucleic acid which contains a complementary segment of nucleic acid. Hybridization techniques are useful where the identification of the presence and location of a particular sequence of nucleic acids is desired. Usually, only a few of the hundreds of bands will be visualized by the probe. Hybridization techniques are specific and usually quite sensitive. Probes using radioactive isotopes as labels are detectable at about 1 pg/mm.sup.2 band cross-section. Since the probe is so specific, there is very little background interference, which contributes significantly to the sensitivity of the technique. However, the use of radioisotopes as probes pose a significant threat to the health of workers and to the environment.
In contrast, the direct visualization techniques usually mark all of the bands in the gel and not just a specific few. In addition, there is some deposition of the visualizing material, such as silver, into the background gel. By far, silver staining is the preferred method of direct visualization of nucleic acids in gels. The methods for silver staining are derived either from histological procedures using ammoniacal silver solutions or from photochemical procedures in which silver ions bind to the nucleic acid bases and are then selectively reduced by chemical agents or light.
Generally, silver staining methods are not as sensitive as the radioisotopic methods since the gel will usually take up some of the silver ions giving a lowered contrast between the gel background and the bands of nucleic acids. Some prior silver staining methods have been made as sensitive as radioisotope methods but they are complex, time consuming and require the preparation of several moderately unstable solutions immediately prior to use. These complications greatly decrease the usability of the most sensitive silver staining methods.
Accordingly, it is an object of the present invention to provide a method for visualizing nucleic acids in polyacrylamide gels that has substantially the same sensitivity and ease of use as radioisotope markers without the inherent radiation risks to technicians or the environment.
Additionally, it is an object of the present invention to provide a method for visualizing nucleic acids in polyacrylamide gels using a silver staining technique that is neither as complex nor as time consuming as methods in the prior art while having the sensitivity of those methods using radioisotopes.